DESCRIPTION: The Specific Aims: 1) comparison of 2-disulfide analogs of insulin and IGF-1, 2) characterization of a 1-disulfide IGF-1 folding intermediate and a minimalist peptide model, 3) comparative 15N relaxation studies on IGF-1 analogs, 4) mutagenesis studies aimed at producing molecules that fold with the characteristics of either insulin or IGF-1, and 5) characterization of the in vivo folding pathway. The overall experimental plan is based on the observation that two proteins, insulin and IGF-1, that are highly similar to each other in their native, folded, disulfide-bonded states, exhibit significantly different folding properties. In particular, oxidative refolding under thermodynamic control yields a single (native) product for insulin, but yields two products (native, and one in which two of the three disulfides have swapped pairings) for IGF-1. This differential property is the basis on which Dr. Weiss proposes to identify factors responsible for on-pathway and off-pathway oxidative folding.